Oligo RNA is useful as RNA probes for gene analysis, pharmaceutical RNA materials (antisense RNA, ribozymes, RNA for control of gene expression through RNAi), artificial enzymes, and aptamers. As reagents for use in the preparation of the oligo RNA, phosphoramidite compounds substituted at the 2′-hydroxyl group of the ribose with the 2-cyanoethoxymethyl (CEM) group, which can be removed under neutral conditions, have been reported (Ohgi et al., Organic Letters, Vol. 7, 3477 (2005); WO 2006/022323 A1).
The production of the phosphoramidite compounds includes a step for introducing CEM as a protecting group at the 2′-hydroxyl group of the ribose. This step has so far been performed by reacting a ribonucleic acid derivative (raw material) protected with a silicon protecting group (for example, tetraisopropyldisiloxan-1,3-diyl) at the 3′- and 5′-hydroxyl groups of the ribose, with methylthiomethyl 2-cyanoethyl ether as an alkylating reagent and N-iodosuccinimide (NIS) or N-bromosuccinimide (NBS) as a reagent (oxidant) for halogenating the sulfur atom in the alkylating reagent, in the presence of an acid such as trifluoromethanesulfonic acid or silver trifluoromethanesulfonate (WO 2006/022323 A1).
Since the reagent described above for halogenating the sulfur atom in the alkylating reagent and the acids described above, namely NIS, NBS, trifluoromethanesulfonic acid and silver trifluoromethanesulfonate, are highly reactive compounds, the nucleobase in the ribonucleic acid derivative as the raw material compound may also possibly be halogenated, even when the reaction temperature is lowered to about 0° C. Therefore, it is necessary to carry out the reaction at very low temperatures of −50° C. to −40° C., so as to prevent the halogenation of the nucleobase. On a small scale (for example, when the amount of the ribonucleic acid derivative as the raw material compound is about 100 mg to about 2 g), the reaction may sometimes proceed smoothly even when the reaction temperature is around 0° C.
When NIS and NBS are used, byproducts derived from the succinimide are generated. It is very hard to remove these byproducts by extraction procedures. Generally, it is necessary to remove the byproducts by column chromatography, which is unsuitable for large-scale purification.
Furthermore, NIS, NBS, trifluoromethanesulfonic acid, and silver trifluoromethanesulfonate are very expensive reagents and so carry an economic disadvantage.
Therefore, such conventional processes using NIS and NBS as those described above are not suitable for large-scale production of the phosphoramidite compounds.